Mechanistic Insights into Synergistic Phytotoxicity of Imidacloprid and Florasulam in Freeze–Thaw Stressed Rye Seedlings: Antioxidant Disruption and Photosynthetic Impairment

The combined effects of insecticides and herbicides under freeze-thaw cycles, common in high-latitude agriculture but poorly understood, were investigated using rye (<i>Secale cereale L.</i>). Physiological analyses assessed the impacts of imidacloprid (IMI) and florasulam (FLS) under freeze-thaw stress. IMI and FLS synergistically inhibited rye growth (reducing root length, height, and biomass), induced oxidative stress, altered osmoregulation, and reduced photosynthesis. Freeze-thaw conditions exacerbated pesticide damage, inducing more severe growth and photosynthetic inhibition. Notably, combined stress elevated malondialdehyde (MDA), soluble protein (SP), and relative electrolyte conductance (REC) by 23.8%, 10.7%, and 2.27-fold, respectively, while suppressing key antioxidant enzyme activities by 31.5-42.4%. Molecular docking indicated binding of pesticide to antioxidant enzyme and chlorophyll protein active sites via hydrogen bonds. Both the integrated biomarker response (IBR) and partial least-squares path model (PLS-PM) heightened toxicity under combined stress. This study reveals the toxic mechanisms of combined freeze-thaw, insecticide, and herbicide stress on high-latitude crops.